In contrast to the rather large amount of information available concerning the molecular organization of proteins and phospholipids in membranes, relatively little is known about how glycosphingolipids are organized in the cell plasma membrane. Clycosphingolipids are components of mammalian cell surface membranes which represent differentiation and blood group antigens, receptors for hormones, toxins, and microbes and mediators of cell-cell recognition and interaction. Because of the unique features of the proposed experimental system, new information will be obtained about the distribution of glycosphingolipids in membranes. The specific goal of this project is to determine the glycosphingolipid composition of the apical and basolateral domains of the plasma membrane of the Madin Darby canine kidney (MDCK) epithelial cell line. A monolayer of these cells exhibits polarity in structure and function and resembles transporting epithelia. Enveloped viruses obtain their envelopes by budding through the host cell plasma membrane and have lipid compositions reflecting those of the cell plasma membrane. In the case of MDCK monolayers, influenza virus buds from the apical membrane while vesicular stomatitis virus (VSV) buds from the basolateral membrane domain. Thus, each virus represents a vehicle for assaying the lipids of the MDCK membrane domain from which it budded. Our protocol will involve metabolic labeling of MDCK monolayers with radioactive precursors of glycolipids followed by infection with either influenza or VSV. Budding viruses will be harvested, purified, and the glycolipid profiles analyzed by thin layer chromatography and autoradiography. The mechanisms responsible for generating and maintaining the polarized plasma membranes of transporting epithelia are poorly understood. These studies on the distribution of glycolipids in such polarized cells may reveal details of these important mechanisms. An additional long range goal of this approach is to use the MDCK cell system to develop methods that will be of general use for analyzing the glycolipid composition of membrane domains of other polarized epithelial and endothelial cells.